Adeno-associated viruses (AAVs) are nonenveloped, single-stranded DNA viruses of the family Parvoviridae[1] and the genus Dependovirus that are not associated with any known disease and show great potential as gene transfer vectors[2]. AAVs depend on co-infection with a helper virus, such as adenovirus, herpes-virus, or papillomavirus for productive replication[3]. AAV has been considered as a promising vehicle for human gene therapy based on its ability to infect both dividing and non-dividing cells, as well as establish long-term gene expression in vivo without known pathological consequence of infection[4-7]. The AAV type 2 (AAV2) nanoparticles constitutes the first primate AAV to be cloned, and promising results have been obtained with this nano vector in clinical gene transfer, including cystic fibrosis[8], retinal degenerative disorders[9-11] and haemophilia B[12, 13]. 
In recent years, there have been intensive efforts in many laboratories to generate targeted AAV vectors by modifying the cell-binding characteristics of these particles. The primary strategy has been to genetically modify the AAV capsid proteins by insertion of targeting peptide motifs that can direct nano vectors to specific cell types. This method has been successfully employed to retarget AAV to arterial endothelium[14], striated muscles[15], and brain vasculature[16]. However, the major technical challenges in this manipulation include the low production yield, dramatic reduction of vector titer, or significant drop of DNA packaging efficiency[17]. The large-scale genetic engineering modifications of viral capsid may abrogate their infectivity and even alter the innate interactions between viruses and host cells. For this reason, it is necessary to develop a site-selective and non-destructive technique for modifying adeno-associated viruses.
After several years of studying, comprehensive understanding of ribosome translation mechanism of prokaryotic organisms is almost achieved, crystalline and electron microscopic structures of many ribosomes under different functional status have been resolved; and structures of most aminoacyl-tRNA synthases have been obtained. On the basis of these achievements, a technology of expanding genetic code is developing in recent years, in which an amber termination codon (TAG) is used to encode a variety of non-natural amino acids and to perform site-specific incorporation into organisms in vivo. So far, this technology has been successfully used to site-specifically express dozens of non-natural amino acids in proteins of living cells, which endow these proteins with novel physical, chemical and physiological properties. By using this method, non-natural amino acids (including amino acids for affinity labeling and photoisomerization, carbonylated amino acids and glycosylated amino acids) can be incorporated into proteins (L. Wang, et al, (2001), SCIENCE 292:498-500; J. W. Chin, et al, 2002, Journal of the American Chemical Society 124:9026-9027; J. W. Chin, &P. G. Schultz, 2002, ChemBioChem 11:1135-1137). These researches show that it is possible that chemical groups such as carbonyl, alkynyl and azido can be selectively and conventionally into proteins, in which these groups generally can effectively and selectively form stable covalent bonds, which is advantageous for site-specific modification of proteins and improvement of properties of proteins.
However, this technology has not been applied in site-modification of adeno-associated virus yet.